Enzymes are generally important molecules in living systems considering that they are responsible for the catalysis of the synthesis and/or degradation of biomolecules. As used herein, the term “enzyme” means any molecule that catalyzes or promotes a biochemical reaction without itself being changed or consumed by the reaction. Enzymes work by lowering the activation energy for a chemical reaction and are specific for particular reactions. Enzyme specificity is defined by the molecular shape and/or charge at the active site of a substrate and thus, the enzymes and the substrates fit together like compatible puzzle pieces. Enzymes are generally thought to be protein-based, although there is research to suggest that several enzymes may be something other than protein-based (RNA molecules that catalyze its own splicing). As used herein, the term “substrate” means the substance upon which an enzyme acts. Also as used herein, the phrase “active site” means the region or portion of an enzyme that interacts with the substrate. The three dimensional conformation of amino acids at the active site determines the specificity of the substrate binding. Rudin, Norah, Dictionary of Modern Biology, Barron's Educational Series, Inc. (Hauppauge, N.Y.), 1997.
According to International Union of Biochemistry and Molecular Biology (IUBMB), enzymes can be classified into following six major classes: a) Oxidoreductases catalyze oxidation-reduction reactions and include dehydrogenases (lactate dehydrogenase, acyl CoA dehydrogenase), oxidases (glucose oxidase), peroxidases (Horseradish peroxidase), oxygenases (lactate 2-monooxygenase), and reductases (ketoacyl-ACP reductase); b) transferases catalyze C-, N-, P-, S-containing functional group transfers and include kinases (glucokinase, tyrosine kinase), aminotransferases (aspartate and glutamate aminotransferases), and thiolases (β-ketothiolase); c) hydrolases catalyze hydrolysis reactions involving esters, anhydrides, peptide bonds, glycosides and include peptidases (trypsin, carboxypeptidase), glycosidases (α-glucosidase, amylase, maltase), lipases (triacylglycerol lipase), phosphatases (fructose-1,6-bisphosphatase) and esterases (Cholesterol esterase, acetylcholinesterase, phosphodiesterase); d) lyases catalyze elimination (or addition) of groups to form (or break) double bonds and include synthases (citrate synthase), decarboxylases (pyruvate decarboxylase) and dehydratases (fumarase, aldolase); e) isomerases catalyze reactions that alter structure, not composition (optical, geometric, or structural isomers) and include isomerases (glucose-6-phosphate isomerase, ribulose phosphate epimerase) and mutases (phosphoglycerate mutase); and f) ligases catalyze coupling of two compounds along with hydrolysis of a phosphoric anhydride bond and include synthetases (glutamine synthetase), carboxylases (pyruvate carboxylase) and polymerases (DNA polymerase).
Enzyme inhibitors are those molecules or groups of molecules that reversibly or irreversibly inhibit the catalytic activity of enzymes. Enzyme inhibition generally works in one of four ways in a biological system: a) by allosteric inhibition (inhibition carried out early in a biochemical pathway; the final product is bound to a modulator site that closes the active site by changing its shape, and when levels of the final product are reduced, the inhibitor disengages, re-enabling the synthetic pathway); b) by competitive inhibition (the inhibitor competes with the normal substrate for an enzyme's active site, and the level of inhibition is dependent on the relative concentrations of the substrate and the inhibitor); c) by uncompetitive inhibition (the inhibitor combines with the enzyme-substrate complex, preventing the complex from completing the reaction); and d) non-competitive inhibition (the inhibitor prevents dissolution of the enzyme-substrate complex by binding at a modifier site and effecting a deformation of the active site). Rudin, Norah, Dictionary of Modem Biology, Barron's Educational Series, Inc. (Hauppauge, N.Y.), 1997. Enzyme inhibitors can be administered or introduced into a biological system for any suitable and appropriate purpose, including for research purposes, diagnostic purposes or therapeutic purposes.
When enzyme inhibitors are being used for therapeutic purposes, they are usually being administered to halt or moderate the production of a biomolecule or byproduct of the degradation and/or production of a biomolecule that, in its current concentration, can have a detrimental effect on the biological system. Enzyme inhibitors can be natural or synthetic—and the synthetic enzyme inhibitors often resemble the natural enzyme inhibitors from a chemical and/or activity perspective.
Enzyme inhibitors, however, can encounter problems when dealing with a family of enzyme isoforms or isoenzymes. As used herein, the terms “isoform” and “isoenzyme” can be used interchangeably and can be defined as a variant of an enzyme that exists in different structural forms within a single species. Each isoform or isoenzyme has the same substrate specificity, but often has different substrate affinities. The different isoforms or isoenzymes in a particular family of isoforms or isoenzymes all have the same or similar molecular weights but may differ in configuration or charge. Each isoform or isoenzyme of a particular family of isoforms or isoenzymes can be distinguished by appropriate analytical techniques, including electrophoresis and the corresponding detection instrumentation. It may be that one of the isoforms should be inhibited and not any of the other isoforms. And beyond that, it may be that a particular isoform is targeted to be knowingly and selectively inhibited or “switched off”.
Nitric oxide synthases (NOS) catalyze the oxidation of an L-arginine guanidinium nitrogen atom to nitric oxide (NO), a potent biological signaling molecule that mediates a diverse range of physiological processes within the cardiovascular, immune and nervous systems (S. Moncada et. al. 1991. Pharmacol. Review 43, 109; J. L. Dinerman et. al. 1993. Circ. Res. 73, 217 and J. F. Kerwin Jr. et. al. 1995. J. Med. Chem. 38, 4343). Three mammalian NOS isoforms, nNOS (neuronal nitric oxide synthase: a constitutively expressed isoform located in neuronal tissue and involved in neurotransmission and long-term potentiation); eNOS (endothelial nitric oxide synthase: a constitutively expressed isoform which is involved in the regulation of smooth muscle relaxation and vascular tone); and iNOS (inducible nitric oxide synthase: the isoform in activated macrophage cells that plays a key role in normal immune responses by functioning as a cytotoxic agent), share a common modular architecture consisting of the active site heme domain where L-arginine and the essential cofactor, tetrahydrobiopterin, bind. The C-terminus of the heme domain is connected via a calmodulin-binding linker to the FMN/FAD domain, which shuttles electrons from NADPH to the heme domain (O. W. Griffith and D. J. Stuehr, 1995. Ann. Rev. Physiol. 57, 707 and B. S. Masters et al., 1996. FASEB J. 10, 552). The isoforms of NOS share only approximately 50% primary sequence homology, which suggests that they may differ from each other in regulatory aspects; however, there is very high sequence identity across species. (H. Huang et al, 2000. J. Med. Chem., 43, 2938–2945; J-M Hah et al, 2001. J. Med. Chem., 44, 2667; R. B. Silverman et al, 1997. J. Med. Chem., 40, 2813–2817; H. Huang et al, 1999. J. Med. Chem., 42, 3147, which are all incorporated herein in their entirety).
The over and under production of NO contributes to a large number of pathological conditions (S. Moncada et. al. 1991. Pharmacol. Review 43, 109; J. L. Dinerman et. al. 1993. Circ. Res. 73, 217 and J. F. Kerwin Jr. et. al. 1995. J. Med. Chem. 38, 4343). Hyperactivity by iNOS and nNOS leads to locally high, cytotoxic concentrations of NO and has been identified as a cause of a number of human diseases. Chronic neuro-degenerative diseases such as Alzheimer's, Parkinson's, and Huntington's correlate with overproduction of NO (V. L. Dawson and T. M. Dawson, 1998. Prog. Brain Res. 118, 215). In addition, chronic inflammatory diseases such as arthritis (C. O. Bingham 3rd., 2002. J. Rheumatol. Suppl. 65, 3) and colitis (C. H. Cho, 2001. J. Physiol. Paris, 95, 253) are directly linked to NO overproduction. The hypotension experienced during septic shock, the death of insulin producing pancreatic cells and the extensive cell death that occurs following stroke are likewise due to excessive production of NO (9). On the other hand, impaired NO production by eNOS results in endothelial dysfunction which is responsible for disease states such as hypertension (S. Taddei et al, 2001. J. Cardiovasc. Pharmacol. 38, Suppl. 2, S11) and atherosclerosis (B. S. Sachais, 2001. Curr. Atheroscler. Rep. 3, 412; E. A. Rekka and N.C. Chrysselis, 2002. Mini Rev. Med. Chem. 2, 433). As a result, the identification of isoform-selective NOS inhibitors is important in, for example, decreasing the production of NO by nNOS to minimize neuronal degeneration, but not affect the critical role eNOS plays in regulating blood pressure.
Thus, there remains a need in the art for the development of new treatments for inhibiting nNOS or otherwise preventing or deterring NO overproduction.